Hybrid vector having a cytomegalovirus enhancer and myeloproliferative sarcoma virus promoter

ABSTRACT

An expression vector capable of expressing high levels of heterologous proteins having a cytomegalovirus (CMV) enhancer 5′ upstream from a myeloproliferative sarcoma virus (MPSV) promoter.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation of U.S. patent application Ser. No. 10/465,156,filed Jun. 18, 2003, which claims the benefit under 35 U.S.C. 119(e) ofprovisional application No. 60/389,612, filed Jun. 18, 2002, both ofwhich are herein incorporated by reference.

INTRODUCTION

The present invention is related to the construction and utilization ofa DNA plasmid vector, in particular, those hybrid non-retroviral vectorsthat comprise the cytomegalovirus (CMV) enhancer and themyeloproliferative sarcoma virus (MPSV) promoter minus its negativecontrol region. This hybrid sequence promotes the high expression ofcloned genes under its transcriptional control when the vector istransfected into mammalian cell lines. Preferably, the vector alsocomprises other functional sequences to increase expression of thecloned sequence such as the Ig intron sequence, a viral internalribosome entry site (IRES), a leader sequence to allow for secretedprotein expression, and polyadenylation signals. The vector can alsocomprise selectable markers and other features that facilitate thereplication of the vector in mammalian, yeast, and prokaryotic hostcells, thus increasing the stability of the vector in whateverexpression system is being used.

BACKGROUND OF THE INVENTION

The expression of foreign proteins by bacteria, yeast or mammalian celllines has become routine. One type of commonly used means involves theconstruction of virion-plasmid hybrid vectors that possess the capacityto express cloned inserts in mammalian cells. The expression of thecloned gene with such hybrid vectors can occur in a transient,extrachromosomal manner, but higher production is usually obtainedthrough random insertion of the vector into the host cell genome. Thetypical mammalian expression vector will contain regulatory elements,usually in the form of viral promoter or enhancer sequences andcharacterized by a broad host and tissue range, a polylinker sequencefacilitating the insertion of a DNA fragment within the plasmid vector,and the sequences responsible for intron splicing and polyadenylation ofmRNA transcripts. This contiguous region ofpromoter-polylinker-polyadenylation site is commonly referred to as thetranscription unit. Viral promoter and enhancer regions have long beenutilized as regulatory elements for use in mammalian host cells. Forexample, the strength of the CMV enhancer caused it to be a suggestedcomponent in eukaryotic expression vectors upon its discovery (Boshartet al., Cell, 41 (2):521-30 (1985)) and it has been utilized as auniversal cell control element in transgenic mice (Schmidt et al. Mol.Cell. Biol. 10: 4406-4411 (1990)). The MPSV promoter coveys a wide hostcell specificity to the virus including fibroblasts and hematopoieticstem cells (Stocking et al. Proc. Natl. Acad. Sci. USA, 82: 5746-5750(1985)). Accordingly, this promoter has been used to expressheterologous genes in a number of cell types, including skin fibroblasts(Pamer et al., Blood, 73: 438-445 (1989), primary hepatocytes (Ponder etal., Hum. Gene Ther. 2:41-52 (1991), and rodent cells lines and humanfibroblast cell lines (van den Wollenberg, Gene 144: 237-241 (1994)).

Generally, there are two types of expression vectors suitable for use ineukaryotic cells, retrovirally-based systems and virion-plasmid hybridsdescribed above. van den Wollenberg et al. describe a retroviral vectorthat comprises the CMV enhancer genetically engineered within the U3region of the MPSV promoter. However, retroviral vectors havesignificant drawbacks for use in industrial level protein production.First, the level of protein production is severely hampered by theretroviral packaging sequence, a necessary component of such vectors, asit interferes with translational initiation. Second, protein productionis reduced because the transport of retroviral messenger RNA is lessefficient than a standard mRNA and there is competition betweenretroviral packaging and translation. Third, it is impossible to reachthe gene copy numbers routinely achieved by standard vectors with anamplifying selection marker, due to the fact that a retroviral vectorimplants two promoters for each random integration, thus randomlyactivating downstream sequences with deleterious effects to the cell.Fourth, there are serious safety concerns with large-scale production ofretroviral cultures due to random recombination to replicationcompetency. Finally, retrovirally-established cell lines are harder todocument and less efficient to develop since a viral production cellline must first be used to make a master cell bank, then the actualproduction cell line is produced, requiring a second round of analysisand banking. Accordingly, industrial production of protein is notroutinely performed with retroviral vectors.

Thus, the expression of foreign proteins in commercially acceptablequantities remains a challenge. This is especially true in mammaliancell lines. Very often expression of a mammalian protein in a mammaliancell line is required in order to mimic the native form of the proteinin all respects: structure, catalytic activity, immunologicalreactivity, and biological function. Often glycosylation or otherpost-translational modifications are the key to the production of thedesired form of the protein, and bacteria or yeast systems are unable toaccomplish these modifications. Thus, there remains a need for improvedplasmids that promote the production of mammalian proteins incommercially viable quantities within mammalian host systems.

BRIEF SUMMARY OF THE INVENTION

One aspect of the present invention is a non-retroviral expressionvector comprising a cytomegalovirus (CMV) enhancer and amyeloproliferative sarcoma virus (MPSV) promoter. Preferably, the CMVenhancer is located upstream from the 5′ end of the MPSV promoter. Mostpreferably, the CMV enhancer and MPSV promoter construct comprises thepolynucleotide sequence of SEQ ID NO:1.

The vector of the present invention can further comprise at least oneadditional element selected from the group consisting of a consensus Igintron, a tPA pre-proleader sequence, a polio IRES, a Δ CD8 selectionmarker, and a human growth hormone polyA signal sequence. Preferably,the vector comprises a consensus Ig intron, a tPA pre proleadersequence, and a polio IRES. The vector can also comprise a structuralgene, such as prethrombin.

A further aspect of the present invention is a mammalian celltransfected with the vector. The mammalian cell of the present inventionis preferably a CHO cell, and most preferably a CHO of the strain DXB11.The present invention also encompasses a method of producing arecombinant protein comprising transfecting a mammalian host cell withthe vector of the present invention, growing the cells under conditionsthat selectively propagates those cells that have integrated the vectorinto its genome, and growing the cells with the integrated vector underconditions that cause the recombinant protein to be secreted into thecell medium, and isolating the recombinant protein from the cell medium.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a plasmid map containing the MPSV/CMV promoter/enhancer ofthe present invention. Clockwise the plasmid contains the CMV enhancer,the MPSV LTR promoter minus the negative signal sequence, a consensus Igintron, a tPA pre-pro leader, polio IRES, a ΔCD8 selection marker, ahuman growth hormone (hGH) polyA sequence, a dihydrofolate reductase(DHFR) selection cassette with the SV40 promoter/enhancer and SV40polyA, pUC ori, β lactamase selection, yeast CEN/ARS and URA3 selection.This vector has been named pZMP21.

FIG. 2 compares the picograms per cell per day (pg/cell-day) ofprethrombin production for Chinese Hamster Ovary (CHO) cells transfectedwith pZMP20 (CMV promoter/enhancer) or pZMP21 (MPSV promoter/CMVenhancer).

DESCRIPTION OF THE INVENTION

The present invention fills this need by providing for a novelnon-retroviral expression vector, which is able to transfect mammaliancell lines such as Chinese Hamster Ovary Cells (CHO cells) and promotethe production of foreign proteins in unexpectedly high quantities. Theplasmid of the present invention is comprised of a cytomegalovirusenhancer upstream from the 5′ end of a myeloproliferative sarcoma virus(MPSV) promoter. Preferably the MPSV promoter is fused to acytomegalovirus (CMV) enhancer.

1. Overview

SEQ ID NO: 1 shows a CMV enhancer/MPSV LTR promoter construct of thepresent invention. The CMV enhancer extends from nucleotide 1 to andincluding nucleotide 374 of SEQ ID NO: 1. The MPSV LTR promoter extendsfrom nucleotide 375 to and including nucleotide 851.

2. Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally occurring nucleotides (suchas DNA and RNA), or analogs of naturally occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence.

The term “contig” denotes a nucleic acid molecule that has a contiguousstretch of identical or complementary sequence to another nucleic acidmolecule. Contiguous sequences are said to “overlap” a given stretch ofa nucleic acid molecule either in their entirety or along a partialstretch of the nucleic acid molecule.

The term “structural gene” refers to a nucleic acid molecule that istranscribed into messenger RNA (mRNA), which is then translated into asequence of amino acids characteristic of a specific polypeptide.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Linear DNA” denotes non-circular DNA molecules having free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements [DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993)], cyclicAMP response elements (CREs), serum response elements [SREs; Treisman,Seminars in Cancer Biol. 1:47 (1990)], glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF [O'Reilly et al., J. Biol. Chem. 267:19938 (1992)], AP2 [Ye etal., J. Biol. Chem. 269:25728 (1994)], SP1, cAMP response elementbinding protein [CREB; Loeken, Gene Expr. 3:253 (1993)] and octamerfactors [see, in general, Watson et al., eds., Molecular Biology of theGene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), andLemaigre and Rousseau, Biochem. J. 303:1 (1994)]. If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. Repressible promoters are also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity orconfer tissue specific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter or increases the translation of the mRNAproduct that results from transcription driven by the core promoter. Forexample, a regulatory element may contain a nucleotide sequence thatbinds with cellular factors that increases transcription over basallevels or imparts transcription exclusively or preferentially inparticular cells, tissues, or organelles. Other regulatory elementsincrease translation of the mRNA message that results because ofsequences that are now included in the message, such as an IRES (due toincreased ribosome entry) or a poly-A tail (due to increased mRNAstability).

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

An “integrated genetic element” is a segment of DNA that has beenincorporated into a chromosome of a host cell after that element isintroduced into the cell through human manipulation. Within the presentinvention, integrated genetic elements are most commonly derived fromlinearized plasmids that are introduced into the cells byelectroporation or other techniques. Integrated genetic elements arepassed from the original host cell to its progeny.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage that has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance or ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “non-retroviral vector expression vector” is an expression vector thatdoes not contain a polynucleotide sequence encoding a retroviralpackaging element.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. “Integrativetransformants” are recombinant host cells, in which heterologous DNA hasbecome integrated into the genomic DNA of the cells.

The term “secretory signal sequence” denotes a DNA sequence that encodesa peptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure, orgreater than 99% pure. One way to show that a particular proteinpreparation contains an isolated polypeptide is by the appearance of asingle band following sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis of the protein preparation and Coomassie Brilliant Bluestaining of the gel. However, the term “isolated” does not exclude thepresence of the same polypeptide in alternative physical forms, such asdimers or alternatively glycosylated or derivatized forms.

The terms “amino-terminal or N-terminal” and “carboxyl-terminal orC-terminal” are used herein to denote positions within polypeptides.Where the context allows, these terms are used with reference to aparticular sequence or portion of a polypeptide to denote proximity orrelative position. For example, a certain sequence positionedcarboxyl-terminal to a reference sequence within a polypeptide islocated proximal to the carboxyl terminus of the reference sequence, butis not necessarily at the carboxyl terminus of the complete polypeptide.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity ofless than 10⁹ M⁻¹.

“Upstream” and “downstream” are terms used to describe the relativeorientation between two elements present in a nucleotide sequence. Anelement that is “upstream” of another is located in a position closer tothe 5′ end of the sequence (i.e., closer to the end of the molecule thathas a phosphate group attached to the 5′ carbon of the ribose ordeoxyribose backbone if the molecule is linear) than the other element.An element is said to be “downstream” when it is located in a positioncloser to the 3′ end of the sequence (i.e., the end of the molecule thathas an hydroxyl group attached to the 3′ carbon of the ribose ordeoxyribose backbone in the linear molecule) when compared to the otherelement.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately” X, the statedvalue of X will be understood to be accurate to ±10%.

Polynucleotides, generally a cDNA sequence, of the present inventionencode the described polypeptides herein. A cDNA sequence which encodesa polypeptide of the present invention is comprised of a series ofcodons, each amino acid residue of the polypeptide being encoded by acodon and each codon being comprised of three nucleotides. The aminoacid residues are encoded by their respective codons as follows.

Alanine (Ala) is encoded by GCA, GCC, GCG or GCT;

Cysteine (Cys) is encoded by TGC or TGT;

Aspartic acid (Asp) is encoded by GAC or GAT;

Glutamic acid (Glu) is encoded by GAA or GAG;

Phenylalanine (Phe) is encoded by TTC or TTT;

Glycine (Gly) is encoded by GGA, GGC, GGG or GGT;

Histidine (His) is encoded by CAC or CAT;

Isoleucine (Ile) is encoded by ATA, ATC or ATT;

Lysine (Lys) is encoded by AAA, or AAG;

Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT;

Methionine (Met) is encoded by ATG;

Asparagine (Asn) is encoded by AAC or AAT;

Proline (Pro) is encoded by CCA, CCC, CCG or CCT;

Glutamine (Gln) is encoded by CAA or CAG;

Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT;

Serine (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or TCT;

Threonine (Thr) is encoded by ACA, ACC, ACG or ACT;

Valine (Val) is encoded by GTA, GTC, GTG or GTT;

Tryptophan (Trp) is encoded by TGG; and

Tyrosine (Tyr) is encoded by TAC or TAT.

It is to be recognized that according to the present invention, when apolynucleotide is claimed as described herein, it is understood thatwhat is claimed are both the sense strand, the anti-sense strand, andthe DNA as double-stranded having both the sense and anti-sense strandannealed together by their respective hydrogen bonds. Also claimed isthe messenger RNA (mRNA) that encodes the polypeptides of the presidentinvention, and which mRNA is encoded by the cDNA described herein.Messenger RNA (mRNA) will encode a polypeptide using the same codons asthose defined herein, with the exception that each thymine nucleotide(T) is replaced by a uracil nucleotide (U).

3. Detailed Description

The vector of the present invention can be used to produce polypeptideshaving value in industry, therapeutics, diagnostics, or research.Illustrative proteins include antibodies and antibody fragments,receptors, immunomodulators, hormones, and the like. For example, theexpression vector can include a nucleic acid molecule that encodes apharmaceutically active molecule, such as prethrombin, Factor VIIa,proinsulin, insulin, follicle stimulating hormone, tissue typeplasminogen activator, tumor necrosis factor, interleukins (e.g.,interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, andIL-19), colony stimulating factors (e.g., granulocyte-colony stimulatingfactor, and granulocyte macrophage-colony stimulating factor),interferons (e.g., interferons-α, -β, -γ, -ω, -δ, -τ, and -ε), a stemcell growth factor, erythropoietin, and thrombopoietin. Additionalexamples of a protein of interest include an antibody, an antibodyfragment, an anti-idiotype antibody (or, fragment thereof), a chimericantibody, a humanized antibody, an antibody fusion protein, and thelike. An example of such an antibody fusion protein would be a fusion ofthe extracellular portion of the transmembrane activator andCAML-interactor (TACI) protein, such as amino acids 30-110, fused to theFc portion of human IgG1. The Fc portion can be the native sequence, orone that has been mutated to remove the immunoglobulin effectorfunctions. Examples of these mutations include changes at amino acids234, 235, 237, 330 and 331 of the IgG1 Fc sequence.

The vectors of the present invention have been found to produce theseproteins of interest at higher than expected levels. Without being boundby theory, it is anticipated that the greater than average proteinexpression displayed by the vectors of the present invention is due, atleast in part, to the greater than average stability of expressionexhibited by this vector when integrated into the genome of a mammalianhost cell.

The gene of interest can be isolated from genomic or cDNA sequencesusing methods well known to one of ordinary skill or chemicallysynthesized. If chemically synthesized and double stranded DNA isrequired, then each complementary strand is made separately. Theproduction of short genes (60 to 80 base pairs) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. For the production oflonger genes (>300 base pairs), however, special strategies may berequired, because the coupling efficiency of each cycle during chemicalDNA synthesis is seldom 100%. To overcome this problem, synthetic genes(double-stranded) are assembled in modular form from single-strandedfragments that are from 20 to 100 nucleotides in length.

One method for building a synthetic gene requires the initial productionof a set of overlapping, complementary oligonucleotides, each of whichis between 20 to 60 nucleotides long. The sequences of the strands areplanned so that, after annealing, the two end segments of the gene arealigned to give blunt ends. Each internal section of the gene hascomplementary 3′ and 5′ terminal extensions that are designed to basepair precisely with an adjacent section. Thus, after the gene isassembled, the only remaining requirement to complete the process is toseal the nicks along the backbones of the two strands with T4 DNAligase. In addition to the protein coding sequence, synthetic genes canbe designed with terminal sequences that facilitate insertion into arestriction endonuclease sites of a cloning vector and other sequencesshould also be added that contain signals for the proper initiation andtermination of transcription and translation.

An alternative way to prepare a full-size gene is to synthesize aspecified set of overlapping oligonucleotides (40 to 100 nucleotides).After the 3′ and 5′ extensions (6 to 10 nucleotides) are annealed, largegaps still remain, but the base-paired regions are both long enough andstable enough to hold the structure together. The duplex is completedand the gaps filled by enzymatic DNA synthesis with E. coli DNApolymerase I. This enzyme uses the 3′-hydroxyl groups as replicationinitiation points and the single-stranded regions as templates. Afterthe enzymatic synthesis is completed, the nicks are sealed with T4 DNAligase. For larger genes, the complete gene sequence is usuallyassembled from double-stranded fragments that are each put together byjoining four to six overlapping oligonucleotides (20 to 60 base pairseach). If there is a sufficient amount of the double-stranded fragmentsafter each synthesis and annealing step, they are simply joined to oneanother. Otherwise, each fragment is cloned into a vector to amplify theamount of DNA available. In both cases, the double-stranded constructsare sequentially linked to one another to form the entire gene sequence.Each double-stranded fragment and the complete sequence should becharacterized by DNA sequence analysis to verify that the chemicallysynthesized gene has the correct nucleotide sequence. For reviews onpolynucleotide synthesis, see, for example, Glick and Pasternak,Molecular Biotechnology, Principles and Applications of Recombinant DNA(ASM Press 1994), Itakura et al., Annu. Rev. Biochem. 53:323 (1984), andClimie et al., Proc. Nat'l Acad. Sci. USA 87:633 (1990).

Expression vectors that are suitable for production of an amino acidsequence of interest in eukaryotic cells typically contain (1)eukaryotic or viral DNA elements that control initiation and level oftranscription, such as a promoter and an enhancer; (2) DNA elements thatcontrol the processing of transcripts, such as a transcriptiontermination/polyadenylation sequence; and (3) one or more selectablemarker gene(s) and other sequences useful for stable gene expression forall anticipated host cells. Expression vectors can also includenucleotide sequences encoding a secretory sequence that directs theheterologous polypeptide into the secretory pathway of a host cell.

To express a gene of interest or a selectable marker gene, a nucleicacid molecule encoding the amino acid sequence must be operably linkedto regulatory sequences that control transcriptional expression andthen, introduced into a host cell. The vector of the present inventioncomprises the MPSV promoter with the CMV enhancer in a 5′ position tothe promoter. MPSV is a member of the Moloney murine sarcoma virusfamily (Mo-MuSV) and can transform fibroblasts in vitro and causesarcoma in vivo. Additionally, MPSV causes an acute myeloprolerativedisease in adult mice. The mos oncogene, which is a component of thevirus genome, is necessary for the virus' transforming function, but itis sequences specific to its long terminal repeat (LTR) that account forexpanded cell target specificity when compared to Mo-MuSV. Theseadditional cell targets makes the MPSV LTR an attractive promoter formammalian cell line expression. The MPSV LTR is generally defined asnucleotides between −416 to +31 in relation to the transcriptioninitiation site located within the LTR sequences, although othersequences of the MPSV LTR that function as a promoter can also be used.Preferably, the MPSV promoter has the sequence of nucleotides 375 to 851of SEQ ID NO: 1. The MPSV LTR includes sequences that have beenidentified as negatively controlling transcription. Although deletion ofthese sequences proved to have marginal effect on protein production, sothey remain in pZMP21, they can optionally be deleted in the vector ofthe present invention.

The second regulatory element of the present invention is the CMVenhancer and can be generally defined as the nucleotides between −118and -524 5′ of the transcription initiation site of the majorimmediate-early gene of CMV. Preferably, the CMV enhancer has thesequence of nucletodes 1 to 374 of SEQ ID NO: 1. The enhancer functionof this fragment of the viral genome was discovered based on its abilityto produce recombinant viruses when cotransfected with enhancerless SV40viral genome (Boshart et al., Cell, 41(2):521-30 (1985)). For thevectors of the present invention, this sequence, or functionallyfragments thereof, is placed within the vector such that an increase intranscription results when compared to the transcription without thepresence of the CMV enhancer. Preferably, this location is 5′ of theMPSV promoter sequence.

The vector of the present invention can comprise other regulatoryelements that can increase the expression of the recombinant protein ofinterest within mammalian host cells. Among the other regulatoryelements that can be included is the transcription enhancer locatedwithin the intron of an immunoglobulin gene. Particularly preferred is aconsensus Ig intron sequence that comprises sequences that have beenoptimized for use in mammalian host cells such as CHO DXB11. A secondadditional regulatory element is an internal ribosome entry site (IRES),a sequence derived from viral genomes that allows for the translation ofa dicistronic message. Particularly preferred is the IRES derived fromthe polio virus. A third regulatory element is a poly-A signal sequencethat results in the addition of adenosine residues on the end of themRNA message, which increases the message stability. Particularlypreferred is the poly-A signal sequence derived from the human growthhormone (hGH) gene sequence.

Recombinant host cells can be produced that secrete the amino acidsequence of interest into surrounding medium. Accordingly, the presentinvention contemplates expression vectors comprising a nucleotidesequence that encodes a secretory signal sequence, which is also knownas a “signal peptide,” a “leader sequence,” a “prepro sequence,” or a“pre sequence.” The secretory signal sequence is operably linked to agene of interest such that the two sequences are joined in the correctreading frame and positioned to direct the newly synthesized polypeptideof interest into the secretory pathway of the host cell. Secretorysignal sequences are commonly positioned 5′ to the nucleotide sequenceencoding the amino acid sequence of interest, although certain secretorysignal sequences may be positioned elsewhere in the nucleotide sequenceof interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Hollandet al., U.S. Pat. No. 5,143,830). The present invention can utilize atissue plasminogen activator (tPA) pre-proleader derived from thesequence described in U.S. Pat. No. 5,641,655. Mutations have beenintroduced into the pre-proleader so that it is optimized for use withinmammalian expression systems.

Expression vectors can also comprise nucleotide sequences that encode apeptide tag to aid the purification of the polypeptide of interest.Peptide tags that are useful for isolating recombinant polypeptidesinclude polyHistidine tags (which have an affinity for nickel-chelatingresin), c-myc tags, calmodulin binding protein (isolated with calmodulinaffinity chromatography), substance P, the RYIRS tag (which binds withanti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which bindswith anti-FLAG antibodies). See, for example, Luo et al., Arch. Biochem.Biophys. 329:215 (1996), Morganti et al., Biotechnol. Appl. Biochem.23:67 (1996), and Zheng et al., Gene 186:55 (1997). Nucleic acidmolecules encoding such peptide tags are available, for example, fromSigma-Aldrich Corporation (St. Louis, Mo.).

A wide variety of selectable marker genes for use in mammalianexpression vectors are available (see, for example, Kaufman, Meth.Enzymol. 185:487 (1990); Kaufman, Meth. Enzymol. 185:537 (1990)).Selectable marker genes generally confer growth resistance to a chemicalor drug, that allow selection of initial positive transformants inbacterial, yeast, or mammalian host cells. Selectable markers fall intotwo functional categories: recessive and dominant. The recessive markersare usually genes that encode products that are not produced in the hostcells, i.e., host cells that lack the “marker” product or function.Marker genes for thymidine kinase (TK), dihydrofolate reductase (DHFR),adenine phosphoribosyl transferase (APRT), and hypoxanthine-guaninephosphoribosyl transferase (HGPRT) are in this category. (see, forexample, Srivastava and Schlessinger, Gene 103:53 (1991); Romanos etal., “Expression of Cloned Genes in Yeast,” in DNA Cloning 2: ExpressionSystems, 2^(nd) Edition, pages 123-167 (IRL Press 1995); Markie, MethodsMol. Biol. 54:359 (1996); Pfeifer et al., Gene 188:183 (1997); Tuckerand Burke, Gene 199:25 (1997); Hashida-Okado et al., FEBS Letters425:117 (1998)).

Dominant markers include genes that encode products that conferresistance to growth-suppressing compounds (such as antibiotics or otherdrugs) and/or permit growth of the host cells in metabolicallyrestrictive environments. Commonly used markers within this categoryinclude a mutant DHFR gene that confers resistance to methotrexate; thegpt gene for xanthine-guanine phosphoribosyl transferase, which permitshost cell growth in mycophenolic acid/xanthine containing media; and theneo gene for aminoglycoside 3′-phosphotransferase, which can conferresistance to G418, gentamycin, kanamycin, and neomycin. More newlydeveloped markers include resistance to zeocin, bleomycin, blastocidin,and hygromycin (see, e.g., Gatignol et al., Mol. Gen. Genet. 207:342(1987); Drocourt et al., Nucl. Acids Res. 18:4009 (1990)).

The use of selectable markers has been extended beyond isolation ofcells that have incorporated the vector sequences to selection for cellsthat are expressing the recombinant protein at a high level. An exampleof this selection process is co-expression of green fluorescent proteinwith the recombinant protein. The use of autofluorescent proteinsprovides a visual mechanism to assess if host cells are overexpressingrecombinant protein. Similar selection can be performed with a cellsurface protein that can be detected with an antibody (e.g. CD4, CD8,Class I major histocompatibility complex (MHC) protein, etc.).Preferably, the cytoplasmic domain of the cell surface protein has beendeleted, in order to reduce the cytological effect on the host cell ofover-expression of the protein. The expression products of suchselectable marker genes can be used to sort transfected cells fromuntransfected cells by such standard means as FACS sorting or magneticbead separation technology. Selectable marker genes can be cloned orsynthesized using published nucleotide sequences, or marker genes can beobtained commercially.

The present vector preferably utilizes as selectable makers a DHFRcassette with the SV40 promoter/enhancer for use in mammalian hostcells, a CD8 Δ construct (Δ indicating that the sequence encoding thecytoplasmic domain of the protein has been deleted) to determinerecombinant gene expression at the cell surface of mammalian cells, βlactamase for use in bacterial host cells, and URA3 for use in yeasthost cells.

A final common component of expression vectors are sequences thatfacilitate the replication of the vector in mammalian, yeast, andbacterial hosts such as centromeres, origins of replication, chromatinstability sequences, and the like, that increase the stability of thevector in the host system. For example, the vector of present inventioncan comprise the pUC origin of replication for use in bacterial hostcells and the S. cerevisiae CEN/ARS origin of replication for use inyeast host cells. Chromatin elements that may modulate proteinexpression levels and/or stability are: locus control regions (LCR),matrix or scaffold attachment regions (MAR or SAR) or insulators.

Both during and after construction of the expression vector comprisingthe amino acid-encoding sequences of interest, the vector is typicallypropagated in a host cell. Vector propagation can be carried out in aprokaryotic host cell, such as E. coli. Suitable strains of E. coliinclude BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH41, DH5, DH51,DH51F′, DH51MCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105,JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see,for example, Brown (ed.), Molecular Biology Labfax (Academic Press1991)). Standard techniques for propagating vectors in prokaryotic hostsare well-known to those of skill in the art (see, for example, Ausubelet al. (eds.), Short Protocols in Molecular Biology, 3^(rd) Edition(John Wiley & Sons 1995) [“Ausubel 1995”]; Wu et al., Methods in GeneBiotechnology (CRC Press, Inc. 1997)).

Alternatively, vector propagation both during or after vectorconstruction can be carried out in eukaryotic cells, such as yeast.Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311, Kawasaki et al., U.S. Pat.No. 4,931,373, Brake, U.S. Pat. No. 4,870,008, Welch et al., U.S. Pat.No. 5,037,743, and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459 (1986), and Cregg, U.S. Pat. No. 4,882,279.

Ultimately, the amino acid sequence of interest may be expressed in anyprokaryotic or eukaryotic host cell as described above. Preferably,using the vector of the present invention, the amino acid sequence ofinterest is produced by a eukaryotic cell, such as a mammalian cell.Examples of suitable mammalian host cells include African green monkeykidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells(293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570;ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34),Chinese hamster ovary cells (CHO—K1; ATCC CCL61; CHO DG44; CHO DXB11(Hyclone, Logan, Utah); see also, e.g., Chasin et al., Som. Cell. Molec.Genet. 12:555, 1986)), rat pituitary cells (GH1; ATCC CCL82), HeLa S3cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548)SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650) and murineembryonic cells (NIH-3T3; ATCC CRL 1658). The CHO strain DXB11 is thepreferred host cell for protein production utilizing the vector of thepresent invention.

An expression vector can be introduced into host cells using a varietyof standard techniques including calcium phosphate transfection,liposome-mediated transfection, microprojectile-mediated delivery,electroporation, and the like. Transfected cells can be selected andpropagated to provide recombinant host cells that comprise the gene ofinterest stably integrated in the host cell genome. Standard methods forintroducing nucleic acid molecules into bacterial, yeast, insect,mammalian, and plant cells are provided, for example, by Ausubel (1995).General methods for expressing and recovering foreign protein producedby a mammalian cell system are provided by, for example, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163 (Wiley-Liss, Inc. 1996).

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLE 1

Construction of MPSV Promoter and pZMP21

The MPSV LTR promoter was constructed synthetically by assemblingoligonucleotides in sets of four using PCR.

First the oligos were assembled in pairs by PCR:SEQ ID NOs: 4+5, 6+7,8+9, 10+11, 12+13, 14+15. Then the pairs were assembled into three setsof four oligos SEQ ID NOS: 4+5 and 6+7, with oligos SEQ ID NOs: 4 and 7as primers, 8+9 and 10+11 with oligos 8 and 11 as primers, and 12+13 and14+15 with oligos 12 and 15 as primers in PCR reactions. When the threePCR fragments were assembled a smaller than expected product wasobserved. A new primer, 16, was made to get around the internal repeatthat lead to this deletion. The product of 4+7 was extended with primers4 and 16 to make a better overlap with the product of 8+15.4+16 and 8+15were assembled with primers 4 and 15 by PCR to make a full lengthproduct.

The PCR reactions were run as follows: to a 100 μl final volume wasadded, 10 μl 10×Taq polymerase Reaction Buffer (Perkin Elmer), 8 μl of2.5 mM dNTPs, 78 μl dH₂O, 2 μl each of a 20 mM stock solution of the twoprimers described above, and taq polymerase (2.5 units, LifeTechnology). An equal volume of mineral oil was added and the reactionwas heated to 94° C. for 2 minutes, followed by 25 cycles at 94° C. for30 seconds, 45° C. for 30 seconds, 72° C. for 30 seconds followed by a 5minute extension at 72° C. In the case of the first stage of assemblythe primers were also the templates of the reaction. For the latersteps, 10 μl of PCR product was used as template for the each level ofassembly.

Ten μl of the 100 μl PCR reaction is run on a 1.0% agarose gel with1×TBE buffer for analysis. The remaining 90 μl of PCR reaction isprecipitated with the addition of 5 μl 1 M NaCl and 250 μl of absoluteethanol. The plasmid pZMP20 which has been cut with NheI is used forrecombination with the PCR fragment. Plasmid pZMP20 was constructed frompZP9 (deposited at the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209, and is designated No.98668) with the yeast genetic elements taken from pRS316 (deposited atthe American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209, and designated No. 77145), an IRES elementfrom poliovirus, and the extracellular domain of CD8, truncated at thecarboxyl terminal end of the transmembrane domain. pZMP20 is a mammalianexpression vector containing an expression cassette having thecytomegalovirus immediate early promoter, immunoglobulin signal peptideintron, multiple restriction sites for insertion of coding sequences, astop codon and a human growth hormone terminator. The plasmid also hasan E. coli origin of replication, a mammalian selectable markerexpression unit having an SV40 promoter, enhancer and origin ofreplication, a DHFR gene, the SV40 terminator, as well as the URA3 andCEN-ARS sequences required for selection and replication in S.cerevisiae.

One hundred microliters of competent yeast cells (S. cerevisiae) areindependently combined with 10 μl of the various DNA mixtures from aboveand transferred to a 0.2 cm electroporation cuvette. The yeast/DNAmixtures are electropulsed at 0.75 kV (5 kV/cm), ∞ ohms, 25 μF. To eachcuvette is added 600 μl of 1.2 M sorbitol and the yeast is plated in two300 μl aliquots onto two URA-D plates and incubated at 30° C. Afterabout 48 hours, the Ura+ yeast transformants from a single plate areresuspended in 1 ml H₂O and spun briefly to pellet the yeast cells. Thecell pellet is resuspended in 1 ml of lysis buffer (2% Triton X-100, 1%SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture is added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase is transferred to a fresh tube, and theDNA precipitated with 600 μl ethanol (EtOH), followed by centrifugationfor 10 minutes at 4° C. The DNA pellet is resuspended in 10 μl H₂O.

Transformation of electrocompetent E. coli cells (DH10B, GibcoBRL) isdone with 0.5-2 ml yeast DNA prep and 40 ul of DH10B cells. The cellsare electropulsed at 1.7 kV, 25 μF and 400 ohms. Followingelectroporation, 1 ml SOC (2% Bacto' Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mMMgSO4, 20 mM glucose) is plated in 250 μl aliquots on four LB AMP plates(LB broth (Lennox), 1.8% Bacto Agar (Difco), 100 mg/L Ampicillin).

Individual clones harboring the correct construct are identified byrestriction digest to verify the presence of the MPSV promoter and toconfirm that the various DNA sequences have been joined correctly to oneanother. The insert of positive clones are subjected to sequenceanalysis. Larger scale plasmid DNA is isolated using the Qiagen Maxi kit(Qiagen) according to manufacturer's instruction. pZMP21 was depositedon Jun. 17, 2003 at the American Type Culture Collection (ATCC) 10801University Boulevard, Manassas, Va. 20110-2209, designated as ATCC #PTA-5266.

EXAMPLE 2

Construction of Prethrombin Expression Vectors

An expression plasmid containing all or part of a polynucleotideencoding prethrombin is constructed via homologous recombination. Afragment of prethrombin cDNA is isolated using PCR that includes thepolynucleotide sequence from nucleotide 1 to nucleotide 1380 of SEQ IDNO: 15 with flanking regions at the 5′ and 3′ ends corresponding to thevectors sequences flanking the prethrombin insertion point. The primersfor PCR each include from 5′ to 3′ end: 40 bp of flanking sequence fromthe vector and 17 bp corresponding to the amino and carboxyl terminifrom the open reading frame of prethrombin.

Ten μl of the 100 μl PCR reaction is run on a 0.8% LMP agarose gel(Seaplaque GTG) with 1×TBE buffer for analysis. The remaining 90 μl ofPCR reaction is precipitated with the addition of 5 μl 1 M NaCl and 250μl of absolute ethanol. The plasmids pZMP20 and pZMP21, described in theprevious example, which were cut with BglII were used for recombinationwith the PCR fragment.

One hundred microliters of competent yeast cells (S. cerevisiae) areindependently combined with 10 μl of the various DNA mixtures from aboveand transferred to a 0.2 cm electroporation cuvette. The yeast/DNAmixtures are electropulsed at 0.75 kV (5 kV/cm), ∞ ohms, 25 μF. To eachcuvette is added 600 μl of 1.2 M sorbitol and the yeast is plated in two300 μl aliquots onto two URA-D plates and incubated at 30° C. Afterabout 48 hours, the Ura+ yeast transformants from a single plate areresuspended in 1 ml H₂O and spun briefly to pellet the yeast cells. Thecell pellet is resuspended in 1 ml of lysis buffer (2% Triton X-100, 1%SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture is added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase is transferred to a fresh tube, and theDNA precipitated with 600 μl ethanol (EtOH), followed by centrifugationfor 10 minutes at 4° C. The DNA pellet is resuspended in 10 μl H₂O.

Transformation of electrocompetent E. coli cells (DH10B, Invitrogen) isdone with 0.5-2 ml yeast DNA prep and 40 ul of DH10B cells. The cellsare electropulsed at 1.7 kV, 25 μF and 400 ohms. Followingelectroporation, 1 ml SOC (2% Bacto' Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mMMgSO4, 20 mM glucose) is plated in 250 μl aliquots on four LB AMP plates(LB broth (Lennox), 1.8% Bacto Agar (Difco), 100 mg/L Ampicillin).

Individual clones harboring the correct expression construct forprethrombin are identified by restriction digest to verify the presenceof the prethrombin insert and to confirm that the various DNA sequenceshave been joined correctly to one another. The insert of positive clonesare subjected to sequence analysis. Larger scale plasmid DNA is isolatedusing the Qiagen Maxi kit (Qiagen) according to manufacturer'sinstruction.

EXAMPLE 3

Expression of Prethrombin in Protein-Free, Suspension-Adapted CHO Cells

Serum-free, suspension-adapted CHO DG44 cells were electroporated withtwo of the plasmids described above: pZMP21-prethrombin and the controlplasmid, pZMP20-prethrombin, by the following method. The plasmids werelinearized by digestion with PvuI, precipitated with sodium acetate andethanol then rinsed with 70% ethanol and dried. The pellets wereresuspended at a concentration of 200 μg/100 μl per electroporation inPFCHO medium supplemented with 4 mM L-Glut, 1% Hypoxanthine/Thymidine,1% vitamins, and 1% Na pyruvate (Invitrogen). Cells, growing at logphase, were pelleted and resuspended at 5E6/800 μl per electroporationreaction. The electroporation was performed in a BioRad GenePulser IIwith Capacitance extender (BioRad, Hercules, Calif.), at 300 v and 950μFd in 4 mm cuvettes. The cells were suspended in 25 ml of the mediumdescribed above in 125 mL shake flasks and put on shakers in cellculture incubators at 37° C., at 80 rpm for 24 h to recover. The cellswere then pelleted and resuspended at 2.5E5 in selective medium,consisting of PFCHO supplemented with 4 mM L-Glut, 1% vitamins, 1% NaPyruvate. Cell lines were further cultured in increasing concentrationsof methotrexate up to 1 μM once the cultures were capable of growing inthe absence of hypoxanthine/thymidine supplementation. Once the cultureswere growing actively in selection media and the viability had increasedto over 95%, cultures were established for harvest and analysis ofprotein. Cultures were seeded at 5E5/mL at 25 mL in shake flasks, andallowed to grow for 48 h then harvested. The supernatants were filteredthrough 0.22 μm filters and analyzed by ELISA assay.

The ELISA assay was performed using two polyclonal antibodies: captureantibody, sheep anti-humain prethrombin fragment 2 (Accurate Chemical#20112AP) and detection antibody, sheep anti-human prethrombin-HRPconjugate (Accurate Chemical #20110HP). The coating antibody was dilutedin 0.1 M Na carbonate pH9.6 at 1 μg/mL, dispensed into 96 wells andincubated at 4° C. overnight. The plates were rinsed five times in washbuffer (PBS plus 0.05% Tween) and blocked by incubating twices withSuperBlock (Pierce, Rockford, Ill., #37515) 200 μl/well 5 minutes atroom temperature. The samples and standards were applied to the plate inbinding buffer (PBS, 0.05% Tween, 1 mg/mL BSA) and incubated 1 hour at37° C. The plates were washed five times in wash buffer and detectionantibody diluted to 2 ng/mL in binding buffer. The detection antibodywas applied to the wells and incubated 1 h at 37° C. The plates wererinsed five times with wash buffer and the detection reagent, OPD, wasapplied. OPD was prepared by adding hydrogen peroxide immediately beforeuse according to the manufacturer's instructions (Pierce, Rockford,Ill., #34006), 100 μl added to each well, allowed to develop 10 minutesat RT and stopped with 100 μl per well of 1 N H2SO4. Plates were read at492 nm. The results were calculated via SoftMaxPro. Production rates ofprethrombin by CHO cell pools was calculated by dividing the prethrombintiter by the average number of cells and the number of days in culture.These comparative results are shown in a bar graph in FIG. 2 andindicate that pZMP21-prethrombin produces approximately 3.6 times theamount of recombinant protein as the pZMP20-prethrombin control.

EXAMPLE 4

Construction of zsig37 Expression Vectors

An expression plasmid containing all or part of a polynucleotideencoding zsig37 is constructed via homologous recombination. A fragmentof zsig37 cDNA is isolated using PCR that includes the polynucleotidesequence from nucleotide 1 to nucleotide 873 of SEQ ID NO: 16 withflanking regions at the 5′ and 3′ ends corresponding to the vectorssequences flanking the zsig37 insertion point. The primers for PCR eachinclude from 5′ to 3′ end: 40 bp of flanking sequence from the vectorand 17 bp corresponding to the amino and carboxyl termini from the openreading frame of zsig37.

Ten μl of the 100 μl PCR reaction is run on a 0.8% LMP agarose gel(Seaplaque GTG) with 1×TBE buffer for analysis. The remaining 90 μl ofPCR reaction is precipitated with the addition of 5 μl 1 M NaCl and 250μl of absolute ethanol. The plasmids pZMP20 and pZMP21, described in theprevious example, which were cut with BglII were used for recombinationwith the PCR fragment.

One hundred microliters of competent yeast cells (S. cerevisiae) areindependently combined with 10 μl of the various DNA mixtures from aboveand transferred to a 0.2 cm electroporation cuvette. The yeast/DNAmixtures are electropulsed at 0.75 kV (5 kV/cm), ∞ ohms, 25 μF. To eachcuvette is added 600 μl of 1.2 M sorbitol and the yeast is plated in two300 μl aliquots onto two URA-D plates and incubated at 30° C. Afterabout 48 hours, the Ura+ yeast transformants from a single plate areresuspended in 1 ml H₂O and spun briefly to pellet the yeast cells. Thecell pellet is resuspended in 1 ml of lysis buffer (2% Triton X-100, 1%SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture is added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase is transferred to a fresh tube, and theDNA precipitated with 600 μl ethanol (EtOH), followed by centrifugationfor 10 minutes at 4° C. The DNA pellet is resuspended in 10 μl H₂O.

Transformation of electrocompetent E. coli cells (DH10B, Invitrogen) isdone with 0.5-2 ml yeast DNA prep and 40 ul of DH10B cells. The cellsare electropulsed at 1.7 kV, 25 μF and 400 ohms. Followingelectroporation, 1 ml SOC (2% Bacto' Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mMMgSO4, 20 mM glucose) is plated in 250 μl aliquots on four LB AMP plates(LB broth (Lennox), 1.8% Bacto Agar (Difco), 100 mg/L Ampicillin).

Individual clones harboring the correct expression construct for zsig37are identified by restriction digest to verify the presence of thezsig37 insert and to confirm that the various DNA sequences have beenjoined correctly to one another. The insert of positive clones aresubjected to sequence analysis. Larger scale plasmid DNA is isolatedusing the Qiagen Maxi kit (Qiagen) according to manufacturer'sinstruction.

EXAMPLE 5 Analysis of the Stability of Production of zsig37 by CellsTransfected with MPSV vs. CMV Expression Vectors

Serum-free, suspension-adapted CHO DG44 cells are electroporated withthe plasmids described above, by the following method. The plasmids arelinearized by digestion with PvuI, precipitated with sodium acetate andethanol then rinsed with 70% ethanol and dried. The pellets areresuspended at a concentration of 200 μg/100 μl per electroporation inPFCHO medium supplemented with 4 mM L-Glut, 1% Hypoxanthine/Thymidine,1% vitamins, and 1% Na pyruvate (Invitrogen). Cells, growing at logphase, are pelleted and resuspended at 5E6/800 μl per electroporationreaction. The electroporation is performed in, at 300 v and 950 μFd in 4mm cuvettes. The cells are suspended in 25 ml of the medium describedabove in 125 mL shake flasks and put on shakers in cell cultureincubators at 37° C., at 80 rpm for 24 h to recover. The cells are thenpelleted and resuspended at 2.5E5 in selective medium, consisting ofPFCHO supplemented with 4 mM L-Glut, 1% vitamins, 1% Na Pyruvate. Celllines are further cultured in increasing concentrations of methotrexateup to 1 μM once the cultures are capable of growing in the absence ofhypoxanthine/thymidine supplementation. Once the cultures are growingactively in selection media and the viability has increased to over 95%,cultures are established for harvest and analysis of protein. Culturesare passaged over a period of three months and samples are removedweekly for analysis by ELISA. The supernatants were filtered through0.22 μm filters and analyzed by ELISA assay.

The ELISA assay is performed using two polyclonal antibodies: captureantibody, sheep anti-human zsig37 and detection antibody, sheepanti-human zsig37-HRP conjugate. The coating antibody is diluted in 0.1M Na carbonate pH9.6 at 1 μg/mL, dispensed into 96 wells and incubatedat 4° C. overnight. The plates are rinsed five times in wash buffer (PBSplus 0.05% Tween) and blocked by incubating twices with SuperBlock(Pierce, Rockford, Ill., #37515) 200 μl/well 5 minutes at roomtemperature. The samples and standards are applied to the plate inbinding buffer (PBS, 0.05% Tween, 1 mg/mL BSA) and incubated 1 hour at37° C. The plates are washed five times in wash buffer and detectionantibody diluted to 2 ng/mL in binding buffer. The detection antibody isapplied to the wells and incubated 1 h at 37° C. The plates are rinsedfive times with wash buffer and the detection reagent, OPD, was applied.OPD is prepared by adding hydrogen peroxide immediately before useaccording to the manufacturer's instructions (Pierce, Rockford, Ill.,#34006), 100 μl added to each well, allowed to develop 10 minutes at RTand stopped with 100 μl per well of 1 N H2SO4. Plates are read at 492nm. The results are calculated via SoftMaxPro. Production rates ofzsig37 by CHO cell pools is calculated by dividing the zsig37 titer bythe average number of cells and the number of days in culture. Thelevels of productivity as a function of time are calculated for the twocultures for comparison.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A non-retroviral expression vector comprising a cytomegalovirus (CMV)enhancer and a myeloproliferative sarcoma virus (MPSV) promoter.
 2. Thevector of claim 1 wherein the CMV enhancer is located upstream from the5′ end of the MPSV promoter.
 3. The vector of claim 2 wherein the CMVenhancer and MPSV promoter comprises the polynucleotide sequence of SEQID NO:1.
 4. The vector of claim 1 that further comprises at least oneadditional element selected from the group consisting of a consensus Igintron, a tPA pre-proleader sequence, a polio IRES, a Δ CD8 selectionmarker, and a human growth hormone polyA signal sequence.
 5. The vectorof claim 1 that further comprises a consensus Ig intron, a tPApre-porleader sequence, and a polio IRES.
 6. The vector of claim 2 thatfurther comprises a consensus Ig intron, a tPA pre-proleader sequence,and a polio IRES.
 7. The vector of claim 3 that further comprises aconsensus Ig intron, a tPA pre-proleader sequence, and a polio IRES. 8.The vector of claim 7 further comprising a structural gene such that thegene is operably linked to the CMV enhancer and MPSV promoter.
 9. Thevector pZMP21 as deposited with the ATCC, having the reference numberATCC PTA-5266.
 10. A mammalian cell transfected with the vector ofclaim
 1. 11. The mammalian cell of claim 10 wherein the CMV enhancer andthe MPSV promoter comprises the polynucleotide sequence of SEQ ID NO: 1.12. The mammalian cell of claim 11 wherein the cell is a CHO cell. 13.The mammalian cell of claim 12 wherein the CHO cell is of strain DXB11.14. A method of producing a recombinant protein comprising a.transfecting a mammalian host cell with the vector of claim 1; b.growing the cells under conditions that selectively propagates thosecells that have integrated the vector of claim 1 into its genome; c.growing the cells of step b) under conditions that cause the recombinantprotein to be secreted into the cell medium; d. isolating therecombinant protein from the cell medium.
 15. The method of claim 14wherein the transfection occurs by electroporation.
 16. The method ofclaim 14 wherein the conditions that selectively propagates cells thathave integrated the vector of claim 1 into its genome comprises growingthe cells in the presence of methotrexate.
 17. A method of producing arecombinant protein comprising a. randomly integrating the vector ofclaim 8 into the genome of CHO cells; b. growing the cells in thepresence of increasing concentrations of methotrexate; c. isolatingcells from step b) and growing under conditions such that the CHO cellsproduce the recombinant protein into the culture medium; d. isolatingthe recombinant protein from the culture medium.
 18. The method of claim17 wherein the CHO cells are of the strain DXB11.